1. Bioengineering murine mastocytoma cells to produce anticoagulant heparin
- Author
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Gasimli, Leyla, Glass, Charles A, Datta, Payel, Yang, Bo, Li, Guoyun, Gemmill, Trent R, Baik, Jong Youn, Sharfstein, Susan T, Esko, Jeffrey D, and Linhardt, Robert J
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Hematology ,Animals ,Anticoagulants ,Biotechnology ,Carbohydrate Conformation ,Cell Line ,Tumor ,Heparin ,Mastocytoma ,Mice ,Protein Engineering ,Recombinant Proteins ,Sulfotransferases ,heparan sulfate ,heparin ,mastocytoma ,proteoglycan ,sulfotransferase ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biochemistry and cell biology - Abstract
Heparin (HP), an important anticoagulant polysaccharide, is produced in a complex biosynthetic pathway in connective tissue-type mast cells. Both the structure and size of HP are critical factors determining the anticoagulation activity. A murine mastocytoma (MST) cell line was used as a model system to gain insight into this pathway. As reported, MST cells produce a highly sulfated HP-like polysaccharide that lacks anticoagulant activity (Montgomery RI, Lidholt K, Flay NW, Liang J, Vertel B, Lindahl U, Esko JD. 1992. Stable heparin-producing cell lines derived from the Furth murine mastocytoma. Proc Natl Acad Sci USA 89:11327-11331). Here, we show that transfection of MST cells with a retroviral vector containing heparan sulfate 3-O-sulfotransferase-1 (Hs3st1) restores anticoagulant activity. The MST lines express N-acetylglucosamine N-deacetylase/N-sulfotransferase-1, uronosyl 2-O-sulfotransferase and glucosaminyl 6-O-sulfotransferase-1, which are sufficient to make the highly sulfated HP. Overexpression of Hs3st1 in MST-10H cells resulted in a change in the composition of heparan sulfate (HS)/HP and CS/dermatan sulfate (DS) glycosaminoglycans. The cell-associated HS/HP closely resembles HP with 3-O-sulfo group-containing glucosamine residues and shows anticoagulant activity. This study contributes toward a better understanding of the HP biosynthetic pathway with the goal of providing tools to better control the biosynthesis of HP chains with different structures and activities.
- Published
- 2014